Films based on kefiran, an exopolysaccharide obtained from kefir grain: Development and characterization PDF

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Food Hydrocolloids 23 (2009) 684–690 Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd Films based on kefiran, an exopolysaccharide obtained from kefir grain: Development and characterization Judith Araceli Piermaria a, b, Adriana Pinotti a,...

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Food Hydrocolloids 23 (2009) 684–690

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Films based on kefiran, an exopolysaccharide obtained from kefir grain: Development and characterization Judith Araceli Piermaria a, b, Adriana Pinotti a, c, Marı´a Alejandra Garcia a, Analı´a Graciela Abraham a, b, * a ´n y Desarrollo en Criotecnologı´a de Alimentos CIDCA (CCT-CONICET-La Plata – Facultad de Ciencias Exactas, UNLP), 47 y 116, Centro de Investigacio La Plata (1900), Buenos Aires, Argentina b´ Area Bioquı´mica y Control de Alimentos, Facultad de Ciencias Exactas, UNLP, 47 y 115, La Plata (1900), Buenos Aires, Argentina c Facultad de Ingenierı´a, UNLP, La Plata (1900), Buenos Aires, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 December 2007 Accepted 9 May 2008

Kefiran, an exopolysaccharide produced by microorganisms present in the kefir grains, is a glucogalactan that has several health promoting properties. In the present work, the ability of kefiran to form films and the effect of glycerol addition at different concentrations on film properties was evaluated. Kefiran was able to form films at concentrations ranging from 5 to 10 g/kg. The concentration 10 g/kg was selected because the films were easily removed from the plate. All film-forming solutions exhibited a pseudoplastic behavior; glycerol addition did not modify the solution rheological properties. Kefiran films exhibited differential solubility characteristics, at the different assayed temperatures. These films exhibited good water vapor barrier properties and the addition of 25 g of glycerol per 100 g of polysaccharide allowed the optimum value of 4.09  1011 g/m s Pa to be obtained. Films without glycerol were brittle and rigid since they showed high elastic modulus and tensile strength values and low deformation at break. Glycerol addition led to extremely high elongation values, allowing flexibilities comparable to those of synthetic materials. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Kefiran Kefir Lactic acid bacteria Edible films Glycerol Water vapor barrier and mechanical properties

1. Introduction Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) and polysaccharides isolated from them offer an alternative source of microbial polysaccharides for use in food formulations (Laws, Gu, & Marshall, 2001; Ruas-Madiedo & de los Reyes-Gavila´n, 2005). Exopolysaccharides from LAB have not yet been exploited industrially as food additives, mainly because of the low production levels compared with those produced by gram-negative bacteria such as Xantomonas campestris. For commercial application, exopolysaccharides must be produced at high levels, in low cost media and the isolation procedure must be easy and with high yields. Taking into account these considerations exopolysaccharide from kefir grains might be an affordable alternative (Rimada & Abraham, 2006). Kefir grains, the starter for obtaining the sour fermented milk kefir, are gelatinous irregular masses, composed of proteins and polysaccharides that contain LAB, acetic acid bacteria and yeasts involved in the fermentation (Abraham & De Antoni,

* Corresponding author. Centro de Investigacio´n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA) (CONICET – Facultad de Ciencias Exactas, UNLP), 47 y 116, La Plata (1900), Buenos Aires, Argentina. Tel./fax: þ54 0221 4254853/4249287/ 4890741. E-mail addresses: [email protected], [email protected] (A.G. Abraham). 0268-005X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2008.05.003

1999; Garrote, Abraham, & De Antoni, 2001). The exopolysaccharide produced by kefir microflora named kefiran, is a heteropolysaccharide containing glucose and galactose (Kooiman, 1968; Micheli, Ucelletti, Palleschi, & Crescenzi, 1999) that can be obtained from the grains at high yield produced in deproteinized whey (Rimada & Abraham, 2001). Kefiran improves viscosity and viscoelastic properties of acid milk gels (Rimada & Abraham, 2006) and itself is also able to form gels at low temperature with interesting viscoelastic properties (Piermaria, de la Canal, & Abraham, 2007). Several health promoting properties of kefiran such as immunomodulation or epithelium protection have been reported, being an additional advantage of this exopolysaccharide (Medrano, Pe´rez, & Abraham, 2008; Vinderola, Perdigo´n, Duarte, Farnworth, & Matar, 2006). Otherwise, the use of protective coatings and suitable packaging by the food industry has become a topic of great interest because of their potentiality for increasing the shelf life of many food products. Nowadays, the largest part of materials used in packaging industries is produced from non-renewable material with the negative environmental consequence. A big effort to extend the shelf life and enhance food quality while reducing packaging waste has encouraged the exploration of new biobased packaging materials, such as edible and biodegradable films from renewable resources (Garcı´a, Martino, & Zaritzky, 1998; Sorrentino, Gorrasi, & Vittoria, 2007). Water-soluble

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polysaccharides such as starch, chitosan, cellulose derivatives, alginate, carrageenan and pectin can form edible films (Ferna´ndez-Cervera et al., 2004; Garcı´a, Pinotti, Martino, & Zaritzky, 2004; Krochta & De Mulder-Johnston, 1997; Lawton, 1996; Nisperos-Carriedo, 1994). Polysaccharide-based films are relative stiff, and therefore, plasticizers are needed to facilitate handling. Water, oligosaccharides, polyols and lipids are different types of plasticizer widely used in hydrocolloid-based films (Ayranci & Tunc, 2001; Donhowe & Fennema, 1993; Kim, Ko, & Park, 2002; Sothornvit & Krochta, 2005). Taking into account kefiran characteristics and the necessity of food industry for the development of multipurpose edible films, the aim of the present work was to evaluate the ability of kefiran to form films and the effect of glycerol on film properties. 2. Materials and methods 2.1. Isolation and purification of kefiran A weighed amount of kefir grains CIDCA AGK1 was treated in boiling water (1:10) for 30 min with discontinuous stirring. The mixture was centrifuged at 10,000g for 20 min at 20  C (Avanti J25 Beckman Coulter Inc. centrifuge, Palo Alto, California). The polysaccharide in the supernatant was precipitated by addition of two volume of cold ethanol and left at 20  C overnight. The mixture was centrifuged at 10,000g for 20 min at 4  C. Pellets were dissolved in hot water and the precipitation procedure was repeated twice (Rimada & Abraham, 2006). The precipitate was finally dissolved in hot distilled water and freeze-dried. The samples were tested for the absence of other sugars or proteins by qualitative thin layer chromatography (TLC) and the Bradford method (Bradford, 1976), respectively. TLC was made on Silica gel G type 60 plates (Merck D-64271 Darmstadt, Germany) using n-propanol–acetic acid–water (70:20:10) as the mobile phase. TLC plates were developed with p-amino benzoic acid 7 g/l and o-phosphoric acid 30 g/l in methanol (Zweig & Sherma, 1978). Bradford and thin layer chromatography reagents were obtained from Sigma (St. Louis, MO 63178, USA). 2.2. Film-forming solution preparation Aqueous solutions of 5, 7.5 and 10 g/kg of kefiran were prepared under continuous agitation to select the polysaccharide concentration. The effect of plasticizer addition was studied on the selected concentration of kefiran solution. Glycerol (J.T. Baker, Me´xico) was added as plasticizer; concentrations tested were 12.5, 25.0, 37.5 and 50.0 g per 100 g of kefiran. 2.3. Rheological characterization of film-forming solutions Rheological characterization was performed on the selected polysaccharide concentration solution; plasticizer effect was also studied. Rotational analyses of the film-forming solutions were performed at 25  C in a Haake ReoStress 600 (Thermo Haake, Karlsruhe, Germany) with a 1 mm gap plate–plate sensor system PP35. Shear stress was determined as a function of shear rate. An acceleration of 4.167 s2 was used to increase shear rate from 0 to 500 s1 and the same but negative acceleration value to decrease shear rate until 0. Rheological behavior was correlated by Ostwald de Waele model:

s ¼ kDn where s is the shear stress (Pa), k is the consistency index (Pa sn), D is the shear rate (s1) and n is the flow index (dimensionless).

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Apparent viscosities (mPa s) were calculated at 300 s1 for each film-forming solution. 2.4. Film preparation Unplasticized films and with different concentrations of glycerol were obtained by casting of 25 g filmogenic solutions into Petri dishes (diameter 8.7 cm). Filmogenic solutions were dried at 40  C in a ventilated oven until constant weight along 6 h. The obtained films were removed from the plate and stored at 20  C and 75% relative humidity (RH) in a controlled room. 2.5. Physicochemical characterization of films Humidity content of the films was determined by measuring the weight loss in them, upon drying in an oven at 105  1  C until constant weight (dry sample weight). Samples were analyzed at least in triplicate and the results were expressed as grams of water per 100 g of sample. Water activity of films was evaluated using an AquaLab Water Activity Meter (Decagon Devices, Inc., Washington) equipment. Film thickness was measured using a digital coating thickness gauge Check Line DCN-900 (New York, USA) for non-conductive materials on non-ferrous substrates. Fifteen values were randomly taken at different locations for each specimen and the mean value was informed. Film transparency was determined following the procedure described by Zhang and Han (2006). Film sample was cut into a rectangle and placed on the internal side of a spectrophotometer cell. The absorbance at 600 nm (A600) was recorded for each sample using a Beckman DU650 (Palo Alto, CA, USA) spectrophotometer. Film transparency was calculated by the equation of Han and Floros (1997) as the ratio between A600 and film thickness and was expressed as (A600/mm). In order to determine solubility, three samples from kefiran films without and with glycerol (25 g/100 g polysaccharide) were weighed (0.00001 g), immersed in 25 ml of deionized water at 20, 37 or 100  C and maintained under constant agitation during 2 h. The mixture was centrifuged at 2000g for 10 min and the supernatant was used to evaluate polysaccharide concentration by the anthrone method (Southgate, 1991). The solubility was calculated as the percentage of total carbohydrates present in the film, which were soluble on the studied conditions. 2.6. Microstructural characterization 2.6.1. Scanning electron microscopy (SEM) For cross-section observations, films were cryofractured by immersion of the samples in liquid nitrogen. The pieces of kefiran films without and with 25 g of glycerol per 100 g of polysaccharide were mounted on aluminum stubs using a double-sided tape. Later, they were gold coated with a layer of 40–50 nm of thickness in the ionizer of metals (Jeol FineCoat Sputter JFC-1100, Jeol Ltd. Akishima, Tokyo, Japan) and observed using a Jeol model JSM 6360 LV scanning electron microscope. All samples were examined at an accelerating voltage of 5 kV. 2.6.2. Crystalline degree determination The crystalline structure of film samples without and with glycerol (25 g/100 g polysaccharide) was analyzed by X-ray diffraction in an X’Pert Pro P Analytical Model PW 3040/60 (The Netherlands). The Cu Ka radiation (1.542 Å) was generated at 40 kV and 30 mA, and the relative intensity was recorded in scattering over an angular range (2q) of 3–60 with step size 0.02. The area of the crystalline peak diffraction (AP) relative to the

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total area of the diffractogram (AT) was determined and the crystallinity degree (CD) was calculated as follows:

CD ð%Þ ¼

AP  100 AT

Samples were analyzed at initial time and after 120 days of conditioning at 75% relative humidity (RH) and 20  2  C.

Table 2 Composition and water activity of films without or with different content of glycerol Water activity (aw)

Glycerol (g/100 g of polysaccharide)

Content (%w/w) Humidity

Kefiran

Glycerol

0 12.5 25.0 37.5 50.0

14.79 16.36 21.69 31.86 36.39

85.21 74.34 62.65 49.55 42.41

0.00 9.29 15.66 18.58 21.20

0.453  0.014 0.481  0.004 0.482  0.007 0.541  0.014 0.556  0.021

2.7. Water vapor barrier properties Water vapor permeability (WVP) tests were conducted using the ASTM method E96 with several modifications as described in a previous work (Mali, Grossman, Garcı´a, Martino, & Zaritzky, 2002). Each film sample was sealed over a circular opening of 0.00181 m2 in a permeation cell that was stored at 20  C in a dessicator. The driving force, expressed as water vapor partial pressure, was 1753.55 Pa. To maintain this driving force corresponding to a 75% RH gradient across the film, anhydrous calcium chloride (0% RH) was placed inside and a sodium chloride saturated solution (75% RH) was used in the dessicator. After steady state conditions were reached (about 2 h), eight weight measurements were made. 2.8. Mechanical properties: tensile and puncture tests Puncture tests were performed using a cylindrical probe 2 mm diameter descending at a constant rate of 1 mm/s. Tests were carried out using 3 cm diameter probes and the force and elongation at breaking point of the film were determined. Tensile tests were performed as described in a previous work (Garcı´a et al., 2004) using a tension grip system A/TG and probes of 6 cm length and 0.7 cm width. Both, tensile or puncture tests, were performed in a texturometer TA.XT2i – Stable Micro Systems (England), each informed value corresponded to at least five determinations. Curves of force (N) as a function of deformation (mm) were automatically recorded by the Texture Expert Exceed software. From these curves, maximum breaking force (N) and deformation at break (extension at the moment of rupture, mm) were obtained. In the case of tensile test, elongation at break (deformation divided by initial sample length and multiplying by 100, %) and tensile strength (MPa) were calculated according to the ASTM D882-91 method (1996). 2.9. Statistical analysis All experiments were performed at least in duplicate, with individually prepared and casted films as replicated experimental units. Systat-software (SYSTAT, Inc., Evanston, IL, USA) version 10.0 was used for multifactor analysis of variance. Differences in the properties of the films were determined by Fisher’s least significant difference (LSD) mean discrimination test, using p < 0.05 as level of significance.

Table 1 Rheological properties of 10 g/kg kefiran film-forming solutions without and with 25 and 50 g of glycerol per 100 g of kefiran Glycerol concentration (g/100 g polysaccharide)

0 25 50

Ostwald de Waele parameters

Apparent viscosity (mPa s)

K (Pa sn)

n

D ¼ 300 s1

0.0313  0.0003 0.0311  0.0003 0.0335  0.0008

0.84905  0.0053 0.8472  0.0029 0.8424  0.0088

13.24  0.01 13.01  0.07 13.61  0.33

3. Results and discussion 3.1. Polysaccharide characterization and concentration selection Kefiran, which was obtained with a high purity grade, contained less than 0.01% of protein expressed per dry matter and did not contain mono and disaccharides. The molecular weight obtained by gel permeation chromatography was higher than 4  106 Da (Piermaria et al., 2007). Kefiran concentration in film-forming solution was selected considering the characteristics of the obtained films such as transparency, plate remotion easiness and flexibility. The thickness of the films increased with polysaccharide concentration used as expected from 1.9  1.2 mm for 5 g/kg to 21.0  1.3 mm for 10 g/kg of kefiran solution and all obtained films were transparent. Films were easily removed from the cast plate, except those obtained from 5 g/ kg solution, probably on account of their low thickness (1.9  1.2 mm). Films formulated with 7.5 g/kg had intermediate thickness values, but these films were still difficult to handle. Thus, considering the previously described characteristics the selected kefiran concentration was 10 g/kg.

3.2. Rheological characterization of film-forming solutions With regard to the rheological characterization of kefiran filmforming solutions, all of them exhibited a pseudoplastic behavior. Ostwald de Waele model fitted satisfactorily the experimental data being the correlation coefficient higher than 0.9999 in all cases. The values of the adjusted parameters, n and K, and apparent viscosities of kefiran film-forming solutions were not modified by glycerol addition as is shown in Table 1. The knowledge of the rheological properties of filmogenic solutions is important since they determine the processing conditions and machinability for film obtention at industrial scale. To apply coating in liquid form onto food products by dipping, brushing or sprying, accurate data on rheological properties of filmforming dispersion are needed (Bertuzzi, Armada, & Gottifredi, 2007). Rheological properties of solution are mainly responsible for the presence of defects in the film matrix (Han & Gennadios, 2005). For example, the homogenization of high viscosities solution is difficult; in consequence films with heterogeneities in composition are obtained. Besides high viscosity solutions prevent air bubble to escape from the film and pores (holes) are consequently formed (Peressini, Bravin, Lapasin, Rizzotti, & Sensidoni, 2003). On the

Table 3 Effect of glycerol addition on water vapor permeability (WVP) of kefiran films Glycerol content (g/100 g polysaccharide)

Thickness (mm)

WVP (g/m s Pa)

0.0 12.5 25.0 37.5 50.0

21.43  0.40 21.70  1.56 21.93  1.89 21.00  1.84 18.86  2.11

5.73  1011  7.31  1012 4.55  1011  3.12  1012 4.09  1011  7.47  1012 5.57  1011  2.79  1012 5.71  1011  2.24  1012

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120

687

5 25ºC

100

Initial Stored

37ºC 4

80

Cristaliinity (%)

Solubility (%)

100ºC

60

40

20

3

2

1

0 0

25.0

Glycerol content (g/ 100g polysaccharide) Fig. 1. Solubility at different temperatures of unplasticized kefiran films and plasticized with 25 g of glycerol per 100 g of polysaccharide.

contrary, low viscosities obtained with dilute solutions lead to very thin films. In the case of coatings, rheological behavior as well as surface tension of the solution are important factors linked to solution spreadability and coating adhesion capacity, and thus, limit the application procedure (Hershko & Nussinovitch, 1988). 3.3. Physicochemical film characterization Humidity content and water activity of films increased with plasticizer concentration up to 25 and 37.5 g of glycerol per 100 g of kefiran, respectively (Table 2). On the other hand, at high glycerol concentrations water could be not only associated into the film structure even though retained due to the hygroscopic character of glycerol. Water is incorporated in an active structural form at low glycerol concentrations thus, both glycerol and water molecules plasticized the film matrix. This fact was also described by Even and Carr (1978) in starch-based films. At high glycerol concentration the humidity content also increases, in consequence kefiran concentration decreases, water is not associated to kefiran and water activity increase. Zhang and Han (2006) also reported high values of moisture content on pe...